The role of lipopolysaccharide and cluster of differentiation-14 in the pathogenesis of lupus nephritis

Abstract

Lupus nephritis (LN) is a common cause of acute kidney injury that can lead to chronic kidney failure. Pathogenesis of LN is complex and involves both genetic and environmental factors. Emerging evidence suggests that the gut microbiota and their components contribute to the development of autoimmune diseases, but the underlying mechanisms remain unclear. Bacterial products from the gut may enter the circulation and induce inflammatory responses at distant sites. Lipopolysaccharide (LPS) is a major component of the outer wall of Gram-negative bacteria and is a potent activator of the innate immune system. Cellular response to LPS starts with the transfer of LPS by LPS binding protein (LBP) to CD14, which then presents LPS to TLR-4/MD-2 complex, triggering signal transduction and pro-inflammatory cytokine release. My project investigated changes in the gut microbiota (gut dysbiosis) in patients and mice with active LN, and the roles of LPS, LBP and CD14 in mediating inflammatory and fibrotic processes in the kidney. Fecal DNA from LN patients was analysis by 16S rRNA sequencing. At the phylum level, progression of LN was associated with decreased Gram-positive bacteria Actinobacteria and Firmicutes and increased Gram-negative bacteria Bacteroides and Proteobacteria. At the family level, active LN was associated with Enterobacteriaceae and Porphyromonadaceae. Serum LPS, LBP and CD14 levels correlated with disease activity. Similar findings were observed in NZB/W F1 mice, and active LN was associated with reduced ZO-1 expression in the colon epithelium and increased gut permeability. Orally-fed LPS-FITC was detected in renal proximal tubules. Compared with controls given PBS, LPS-treated mice showed exacerbation of histological features of active LN, collagen deposition, proteinuria and increased gut permeability. In contrast, antibiotic treatment attenuated abnormalities observed in the kidney and colon, and significantly decreased proteinuria. Kidney specimens from patients and NZB/W F1 mice with active LN showed increased LBP and CD14 expression, predominantly in proximal tubular epithelial cells. HK-2 cells that overexpressed LBP and CD14 were used in in vitro studies. CD14 overexpression increased IL-6 secretion and fibronectin expression under basal conditions, which were further augmented upon stimulation with LPS or endotoxin-free TGF-β1. Increased fibronectin expression was mediated partly through increased phosphorylation of p38 MAPK and PI3K. The effect of CD14 on inflammatory and fibrotic processes in HK-2 cells were corroborated in separate studies with CD14 gene silencing by CRISPR/Cas9. LBP overexpression showed no effect on these processes in HK-2 cells. In summary, the data demonstrated that active LN is associated with gut dysbiosis, and increased translocation of LPS from the gut to the circulation. LPS and CD14, but not LBP, contributed to kidney inflammation and fibrosis mediated through p38 MAPK and PI3K signaling pathways.